Optimization of Archael Enzyme Expression in Saccharomyces cerevisiae

Jason D. Smith and Anne S. Robinson
University of Delaware
Department of Chemical Engineering
Newark, DE 19716
smith@che.udel.edu
(302) 831-6556

 A few decades ago life was discovered that thrived in extremes of temperature (4 and 100^oC), pH (2 and 10), salinity (5M NaCl), and pressure (250atm)¹.  These organisms, termed Archae, were found to be a form of life distinct from both bacteria and eukaryotes.² Because of their inherent stability under extreme conditions, there is an industrial interest in exploiting Archael enzymes for applications such as the pulp and paper industry, hydraulic fracturing of gas and oil wells, and specialty chemicals.³  Unfortunately, Archae are difficult to ferment.  They reach much lower biomass yields than Escherichia coli  and Saccharomyces cerevisiae, reproduction of many of their natural environments is beyond modern fermentation technology, and many Archae are elemental sulfur dependent and produce corrosive hydrogen sulfide as a byproduct.  Our goal is to bypass direct fermentation by creating a high yield Archael enzyme expression system in the yeast Saccharomyces cerevisiae.  Current work involves the optimization of gene copy number of our model gene, Pyrococcus furiosus b-glucosidase.⁴  We are using an integrating vector with tunable copy number, determined by G418 resistance.  Preliminary results from copy number optimization and gene expression will be described.  Future work will entail secretory pathway engineering to create a high yield system.

References:

1.  Adams, M., and Kelly, R. 1995.  Enzymes from Microorganisms in Extreme Environments.  C&EN  73: 32-42. 
2.  Woese, C., Kandler, O., and Wheelis, M.  1990.  Towards a natural system of organisms:  Proposal for the domains: Archae, Bacteria, and Eucarya.  Proc. Natl. Acad. Sci. USA  87: 4576-4579.
3.  Adams, M., Perler, F., and Kelly, R.  1995.  Extremozymes: Expanding the Limits of Biocatalysis.  Bio/technology  13: 662-668.
4.  The Pyrococcus furiosus  b -glucosidase was a generous gift of the Kelly Lab at North Carolina State University.